In most cell types, microtubules are organized by the centrosome, an organelle composed of an orthogonal pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome duplicates precisely once. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the centrosome """"""""matures"""""""" or gradually acquires increased levels of microtubule nucleating capacity and PCM. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the nematode Caenorhabditis elegans to study centrosome duplication and maturation. Specifically, our goals are to identify the factors that regulate these processes and to understand how they function on a molecular level. Over the past few years, we have identified and characterized novel regulators of centrosome size and duplication. All were identified in a screen for factors that genetically interact with the kinase ZYG-1, a conserved upstream regulator of centrosome duplication. Most of our work has focused on SZY-20, a conserved RNA-binding protein that localizes to centrosomes and negatively regulates ZYG-1. We have demonstrated that SZY-20 limits centrosome size and that in its absence centrosomes are enlarged. These centrosomes possess elevated levels of ZYG-1 and PCM components such as SPD-2, SPD-5, and gamma-tubulin. Further, we have found that these enlarged centrosomes nucleate more microtubules than their wild-type counterparts and that the enlarged centrosome phenotype is associated with defects in microtubule-dependent processes. Of particular significance we discovered that two centrosome duplication factors, ZYG-1 and SAS-6 play positive roles in defining centrosome size. While disruption of the SZY-20/ZYG-1-mediated size control mechanism affects PCM levels, centriole structure is unperturbed, indicating that the role of ZYG-1 in controlling centrosome size is separable from its role in centriole duplication. To gain further insight into the mechanism of SZY-20 action, we have utilized a proteomics approach to identify proteins that specifically interact with SZY-20. We have identified a large number of proteins that reproducibly co-immunoprecipitate with SZY-20. Among these interactors are known RNA-binding proteins, and regulators of the microtubule cytoskeleton. To determine which of these proteins may play an important role in regulating the centrosome, we used RNAi to deplete each factor from otherwise wild-type animals and monitored centrosome behavior. Depletion of several factors disrupted various centrosome-associated processes. In particular, depletion of the catalytic subunit of protein phosphatase 2A (PP2A) produced a centrosome duplication defect identical to that of ZYG-1. Similarly, depletion of either PAA-1, the PP2A scaffolding subunit, or SUR-6, a PP2A regulatory subunit, results in a centrosome duplication phenotype. Using classical genetics, we have found that ZYG-1 and PP2A exhibit a strong genetic interaction, indicating that these two factors function closely together to regulate centriole duplication. In addition, we find that PP2A and the centriole duplication factor SAS-5 also exhibit a robust genetic interaction. Consistent with our genetic results, we find that PP2A physically interacts with SAS-5 in vivo, and is required to maintain proper levels of ZYG-1 and SAS-5. We are currently analyzing the effect of PP2A depletion on the recruitment of SAS-5 to sites of centriole assembly. While our data demonstrates that PP2A plays a positive role in centriole assembly, we have also found that protein phosphatase I (PP1) plays a negative role. Mutation of the szy-2 gene suppresses a partial loss of ZYG-1 activity. We cloned szy-2 and found that it encodes a regulatory subunit of protein phosphatase 1 (PP1) named I-2. Our genetic analysis indicates that the szy-2(bs4) mutation reduces the activity of I-2 and that I-2 is normally needed to activate GSP-1, a C. elegans PP1 isoform. We find that loss of GSP-1, like loss of SZY-2 suppresses a zyg-1 mutation, indicating that PP1 normally acts to antagonize ZYG-1. However, loss of I-2 mediated regulation does not affect the level of ZYG-1 at centrosomes, suggesting that it acts downstream of ZYG-1 in the centrosome duplication pathway. Since ZYG-1 is a kinase, PP1 may oppose ZYG-1 activity by acting upon one or more of its substrates. We are currently investigating the effect of I-2 depletion on the phosphorylation status of key centrosome duplication genes. Meanwhile, we have found that szy-6, another zyg-1 suppressor, encodes a second regulator of PP1. We are currently analyzing SZY-6 to see if it acts in a manner similar to that of I-2 and we are raising antibodies to determine where in the cell it normally functions. Finally, this past year we have also continued our studies of SZY-5, another negative regulator of ZYG-1. SZY-5 encodes a zinc-finger protein, and like SZY-20, negatively regulates centrosome size. However, SZY-5 appears to function in a manner distinct from that of SZY-20. Unlike SZY-20, SZY-5 does not localize to centrosomes and does not appear to regulate the centrosome-levels of ZYG-1. SZY-5 is found primarily in small cytoplasmic puncta, a distribution reminiscent of processing bodies (P-bodies), which are sites where mRNA stability and translation are controlled. We have postulated that SZY-5 might control the expression of one or more centrosome duplication factors and have used RT-PCR and quantitative immunostaining to address this possibility. We have found that SZY-5 does not significantly affect the mRNA levels of known centrosome duplication factors. However loss of SZY-5 leads to an increase in the levels of SPD-2 at the centrosome. Our results so far indicate that no other centrosome proteins are affected by loss of SZY-5. We have also found that embryos lacking SZY-5 display defects in osmotic integrity, cell cycle progression, polar body extrusion, cytokinesis, and anaphase spindle positioning. As some of the processes affected by the szy-5 mutation involve centrosome function, we are examining centrosome structure and function in the mutant.
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